The subject matter described herein relates to optical microscopy, and more particularly to systems, apparatus, devices and methods for high-resolution optical microscopy based on structure illumination microscopy procedures.
Modern optical microscopy has been key to advancing understanding of structures and functions in living cells. However, one disadvantage has been its diffraction-limited spatial resolution (resolving only features no smaller than half the wavelength used).
Several techniques have been developed to improve the resolution. Near-field scanning optical microscopy (NSOM) forms high resolution images by scanning a sharp tip in the close vicinity of an object. With the access to the optical near field, the resolution is limited by the sharpness of the tip rather than the conventional diffraction. Stimulated emission depletion (STED) microscope is another technique to overcome the diffraction limit by utilizing stimulated emission to sharpen the fluorescence focal spot. Both techniques require the time-consuming point-by-point scanning process, which limits their use in real-time imaging. Another family of the techniques, such as photoactivatable localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), randomly adsorbed molecule microscopy (RAM), which based on the single molecular localization methods, has been emerged mainly in the last few years as a powerful tool to image at deep sub-wavelength scale. The significant spatial resolution improvement, however, has to come with severe imaging speed deterioration. The time for one frame of image is typically about a few minutes or more.
Another emerging field is the superlens and hyperlens imaging approach. Because this technique is based on a projection imaging system, an advantage, in some implementations, of this approach is its ability to go beyond the conventional diffraction limit without affecting too much of the imaging speed.
Structured illumination microscopy (SIM) is another promising technique that has shown great success in fluorescent microscopy. SIM superposes a light pattern on an object with sub-wavelength features to produce fringe patterns which can be optically measured and processed to form a high-resolution image. Significant spatial resolution improvement can be gained using these techniques but with low imaging speed being a trade-off. Standard or linear SIM generally offers a factor of two (2) resolution improvement because the illumination pattern used is diffraction-limited. Non-linear SIM applied to fluorescence microscopy achieves >2 resolution enhancement by introducing higher harmonics to the illumination pattern through the nonlinear, saturated fluorescence response. This technique, however, entails additional limitations (e.g., sample heating/damage, need to acquire more images) and is generally not applicable to scattering microscopy.